Agent for preventing or improving decline in brain function

ABSTRACT

An object of the invention is to provide an agent for preventing or improving decline in brain function such as decreased perception ability, decreased memory learning ability, decreased thinking ability, decreased concentration, decreased attention, decreased judgment ability, depression, and decreased exercise performance caused thereby. According to the invention, an agent for protecting brain neuronal cells, comprising citrulline or a salt thereof and citicoline or a salt thereof as active ingredients as well as an agent for preventing or improving decline in brain function, comprising citrulline or a salt thereof and citicoline or a salt thereof as active ingredients is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of co-pending U.S. patentapplication Ser. No. 14/435,984, filed on Apr. 15, 2015, which is theU.S. national phase of International Patent Application No.PCT/JP2013/080053, filed Oct. 30, 2013, which claims the benefit ofJapanese Patent Application No. 2012-238542, filed on Oct. 30, 2012,which are incorporated by reference in their entireties herein.

TECHNICAL FIELD

The present invention relates to an agent for preventing or improvingdecline in brain function, comprising citrulline or a salt thereof andciticoline or a salt thereof as active ingredients.

BACKGROUND ART

Brain is the most significant region of central nerve system forinformation transmission via nerves such as motion and perception, andplays an important role in human mental activity such as feelings,emotions, reasoning or the like. Higher brain dysfunctions caused bycerebral trauma, cerebrovascular disorders, cerebritis, hypoxia or thelike include a wide range of defects in memory, attention, executivefunctioning, social behavior and the like, and their characteristicsvary depending on the part of the brain damage. Further, it is alsoreported that decline in brain function can be caused by fatigue(Non-Patent Literature 1), leading to decline in abilities such asmemory learning, attention concentration, judgment or the like.

It is known that ischemic cerebrovascular disease causes brain neuronalcell death, leading to decline in brain function. The cause includesneurovascular damage, endothelial dysfunction, diminished cerebral bloodflow or the like (Non-Patent Literature 2), and it is understood that areduction of normal nitric oxide (NO) production in vascular endothelialcells and diminished cerebral blood flow cause brain neuronal cell deathand decline in brain function. In other words, it is expected that brainneuronal cells can be protected and decline in brain function can beprevented or improved by enhancing NO production in vascular endothelialcells and cerebral blood flow.

Citrulline is one of amino acids present as free form and is not used asa constituent for protein synthesis in vivo. In the body, citrullineserves as a precursor of arginine in arginine biosynthesis or animportant component of NO cycle involved in NO supply. Ingestedcitrulline is mostly converted to arginine in the kidney and producedarginine is efficiently supplied systemically (Non-Patent Literature 3).

Arginine is an amino acid to be a direct substrate of nitric oxide (NO)synthase. Moreover, arginine is an intermediate in the urea cycle in theliver, and plays an important role in detoxication of ammonia producedin the body. NO synthesized from arginine as a substrate exerts avariety of physiological functions for maintaining normal vascularfunctions, including vasodilatation, inhibition of LDL oxidation,inhibition of platelet aggregation, anti-proliferation effect on smoothmuscle cells, antioxidant effect and the like.

It is reported that ingestion of citrulline shows ananti-arteriosclerosis action and improves blood circulation via theproduction of the vasodilator NO (Non-Patent Literature 4), andcitrulline is widely used around US as a food material for producing NOto improve blood circulation. Citrulline is also used in Europe as ananti-fatigue drug in the form of citrulline-malate.

Citicoline is a water-soluble substance that is biosynthesized fromcholine phosphate and cytidine triphosphate in animals, yeasts or thelike. Citicoline is involved in the biosynthesis of phosphatidylcholinewhich is a component of cell membrane, acetylcholine which is aneurotransmitter, or the like. It is also reported that citicoline, onceorally ingested, is degraded into choline and uridine during intestinalabsorption, and citicoline is reconstituted in the brain (Non-PatentLiterature 5).

It is reported that ingestion of citicoline improves prognosis of acutecerebral infarction (Non-Patent Literature 6), cognitive function inAlzheimer's-type dementia (Non-Patent Literature 7) or the like. Owingto these functions, citicoline has been used as a medicine for theimprovement of brain metabolism, impaired consciousness and pancreatitisin Japan and as a food material for the improvement or amelioration ofbrain function in foreign countries.

Until now, there have been reports about improvement of attentionconcentration by oral ingestion of citrulline (Patent Literature 1) andimprovement of memory learning ability by intraperitoneal injection ofarginine (Non-Patent Literature 8). However, there have been no reportsthat a synergistic effect of preventing or improving decline in brainfunction can be obtained by using citrulline or a salt thereof incombination with citicoline or a salt thereof.

CITATION LIST Patent Literature

-   [PTL1] WO 2009/096505

Non Patent Literature

-   [NPL1] Journal of occupational health, 2007, Vol. 49, p. 203-208-   [NPL2] Neuroscience, 1999, Vol. 91, p. 203-210-   [NPL3] Amino Acids, 2005, Vol. 29, p. 177-205-   [NPL4] PNAS, 2005, Vol. 102, p. 13681-13686-   [NPL5] Journal of Neuroscience Research, 1999, Vol. 58-5, p. 697-705-   [NPL6] Stroke, 2002, Vol. 33, p. 2850-2857-   [NPL7] Methods and findings in experimental and clinical    pharmacology, 1999, Vol. 21-9, p. 633-644-   [NPL8] Psychopharmacology, 2003, Vol. 167, p. 291-296

SUMMARY OF INVENTION Technical Problem

An objection of the present invention is to provide an agent forpreventing or improving decline in brain function such as decreasedperception ability, decreased memory learning ability, decreasedthinking ability, decreased concentration, decreased attention,decreased judgment ability, depression, and decreased exerciseperformance caused thereby.

Solution to Problem

The present invention relates to the following (1) to (13):

(1) An agent for protecting brain neuronal cells, comprising citrullineor a salt thereof and citicoline or a salt thereof as activeingredients.

(2) An agent for preventing or improving decline in brain function,comprising citrulline or a salt thereof and citicoline or a salt thereofas active ingredients.

(3) The agent for preventing or improving decline in brain functiondescribed in (2), wherein the decline in brain function is one or moreselected from the group consisting of decreased perception ability,decreased memory learning ability, decreased thinking ability, decreasedconcentration, decreased attention, decreased judgment ability,depression, and decreased exercise performance caused thereby.(4) A method for preventing or improving decline in brain function,wherein citrulline or a salt thereof and citicoline or a salt thereofare orally ingested as active ingredients.(5) A method for preventing or improving decline in brain function,wherein an oral preparation comprising citrulline or a salt thereof andciticoline or a salt thereof as active ingredients is ingested.(6) A method for preventing or improving decline in brain function,wherein citrulline or a salt thereof and citicoline or a salt thereofare orally ingested as active ingredients, provided that the preventionor improvement method does not include any method of surgery, therapy ordiagnosis of humans practiced by medical doctor.(7) A method for preventing or improving decline in brain function,wherein an oral preparation comprising citrulline or a salt thereof andciticoline or a salt thereof as active ingredients is ingested, providedthat the prevention or improvement method does not include any method ofsurgery, therapy or diagnosis of humans practiced by medical doctor.(8) Citrulline or a salt thereof and citicoline or a salt thereof foruse in protecting brain neuronal cells.(9) Citrulline or a salt thereof and citicoline or a salt thereof foruse in preventing or improving decline in brain function.(10) The citrulline or a salt thereof and the citicoline or a saltthereof described in (9), wherein the decline in brain function is oneor more selected from the group consisting of decreased perceptionability, decreased memory learning ability, decreased thinking ability,decreased concentration, decreased attention, decreased judgmentability, depression, and decreased exercise performance caused thereby.(11) Use of citrulline or a salt thereof and citicoline or a saltthereof for the manufacture of an agent for protecting brain neuronalcells.(12) Use of citrulline or a salt thereof and citicoline or a saltthereof for the manufacture of an agent for preventing or improvingdecline in brain function.(13) The use described in (12), wherein the decline in brain function isone or more selected from the group consisting of decreased perceptionability, decreased memory learning ability, decreased thinking ability,decreased concentration, decreased attention, decreased judgmentability, depression, and decreased exercise performance caused thereby.

Advantageous Effects of Invention

According to the present invention, a safe and effective agent forprotecting brain neuronal cells and a safe and effective agent forpreventing or improving decline in brain function, comprising citrullineor a salt thereof and citicoline or a salt thereof as active ingredientscan be provided. According to the agent of the present invention, memorybehavior can be synergistically and effectively improved by usingcitrulline or a salt thereof in combination with citicoline or a saltthereof, and excellent effect as the agent for preventing or improvingdecline in brain function can be obtained.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows spontaneous alternation behavior rate (Alternation) in theY-maze test after administration of each concentration of the sample tomice for 7 days. Mean±standard error. N=4 to 6. There are significantdifferences between different letters.

FIG. 2 shows the time spent in the light compartment (Latency time) inthe passive avoidance test after administration of each concentration ofthe sample to mice for 7 days. Mean±standard error. N=4 to 6. There aresignificant differences (p<0.05) between different letters.

FIG. 3 shows a ratio of the frequency of exploring the familiar andnovel objects in the novel object recognition test after administrationof each concentration of the sample to mice for 7 days. Mean±standarderror. N=4 to 6. ** indicates significant differences (p<0.01) betweenthe familiar and novel objects.

FIG. 4 shows the viability of hippocampal nerve cells afteradministration of each concentration of the sample to mice for 12 days.Mean±standard error. N=8. There are significant differences (p<0.05)between different letters.

DESCRIPTION OF EMBODIMENTS

Citrulline used in the present invention includes L-citrulline andD-citrulline. Preferred is L-citrulline. Citrulline can be obtained bychemical synthesis, fermentation or the like. Citrulline can be alsoobtained by purchasing a commercially available product. A chemicalsynthesis method of citrulline includes the methods described in J.Biol. Chem., 122, 477(1938) and J. Org. Chem., 6, 410(1941).

A method for producing L-citrulline by fermentation includes the methodsdescribed in Japanese Patent Publication Nos. 1978-075387 and1988-068091. In addition, L-citrulline and D-citrulline can also bepurchased from Sigma-Aldrich Co., or the like.

The salts of citrulline include acid addition salts, metal salts,ammonium salts, organic amine addition salts, amino acid addition saltsand the like. The acid addition salt includes inorganic acid salts suchas hydrochloride, sulfate, nitrate, phosphate and the like, and organicacid salts such as acetate, maleate, fumarate, citrate, malate, lactate,α-ketoglutarate, gluconate, caprylate and the like. The metal saltincludes alkali metal salts such as sodium salts, potassium salts andthe like, alkaline earth metal salts such as magnesium salts, calciumsalts and the like, aluminum salts, zinc salts and the like. Theammonium salt includes ammonium salts, tetramethylammonium salts and thelike. The organic amine addition salt includes morpholine salts,piperidine salts and the like. The amino acid addition salt includessalts of glycine, phenylalanine, lysine, aspartic acid, glutamic acidand the like. Among the above-mentioned salts of citrulline, malate ispreferably used. Other salts or two or more salts may be used in anappropriate combination.

Citicoline (citidine-5′-diphosphocholine (CDP-choline)) used in thepresent invention may be those obtained by any production method. Theproduction method for citicoline includes the chemical synthesis(Japanese Patent Publication Nos. 1964-6541, 1967-1384, 1988-6558, andthe like), the enzymatic method using a microorganism such as yeast orthe like (Japanese Patent Publication Nos. 1973-2358, 1973-40757,1973-40758, 1978-109996, 1979-14593, 1988-313594, etc.) or the like.Citicoline can be also purchased from Sigma-Aldrich Co., or the like.

The citicoline comprised in the agent for preventing or improvingdecline in brain function of the present invention may exist as a saltthereof in the agent. The salt of citicoline includes the same salt asthat of citrulline as mentioned above.

In the present invention, a compound involved in the synthesis ofciticoline in vivo, for example, choline, uridine, or the like can bealso used instead of citicoline.

A composition ratio of citrulline or a salt thereof to citicoline or asalt thereof in the agent for preventing or improving decline in brainfunction of the present invention is 1:100 to 100:1, preferably 1:50 to50:1, particularly preferably 10:1 to 1:10 by weight.

As the agent for preventing or improving decline in brain function ofthe present invention, citrulline or a salt thereof and citicoline or asalt thereof can be administered as such, but it is usually preferred toprovide the agent as various kinds of preparations.

The preparations comprise, as active ingredients, citrulline or a saltthereof and citicoline or a salt thereof and further may include anyactive ingredients. The preparations are produced according to anymethods well known in the technical field of pharmaceutics by mixing theactive ingredients with one or more kinds of pharmaceutically acceptablecarriers.

Administration routes of the preparation may include oral administrationand parenteral administration such as intravenous administration,intraperitoneal administration, subcutaneous administration or the like.Preferred is oral administration. The preparation may be administeredeither in the form of oral preparations such as tablets, powders,granules, pills, suspensions, emulsions, infusions/decoctions, capsules,syrups, liquids, elixirs, extracts, tinctures, fluid extracts and thelike, or parenteral preparations such as injections, drops, creams,suppositories and the like. Preferred are oral preparations.

Liquid preparations suitable for oral administration such as syrups canbe prepared by adding water, sugars such as sucrose, sorbitol, fructoseor the like, glycols such as polyethylene glycol, propylene glycol orthe like, oils such as sesame oil, olive oil, soybean oil or the like,antiseptics such as p-hydroxybenzoate ester or the like, paraoxybenzoicacid derivatives such as methyl paraoxybenzoate, preservatives such assodium benzoate or the like, flavors such as strawberry flavor,peppermint or the like, or the like.

For example, tablets, powders, granules, or the like suitable for oraladministration can be prepared by adding excipients such as sugars suchas lactose, white sugar, glucose, sucrose, mannitol, sorbitol or thelike, starch such as potato, wheat, corn or the like, inorganicsubstances such as calcium carbonate, calcium sulfate, sodium hydrogencarbonate, sodium chloride or the like, plant powders such ascrystalline cellulose, licorice powder, powdered gentian or the like,disintegrating agents such as starch, agar, gelatin powder, crystallinecellulose, carmellose sodium, carmellose calcium, calcium carbonate,sodium hydrogen carbonate, sodium alginate or the like, lubricants suchas magnesium stearate, talc, hydrogenated vegetable oil, macrogol,silicone oil or the like, binders such as polyvinyl alcohol,hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carmellose,gelatin, starch paste or the like, surfactants such as fatty acid estersor the like, plasticizers such as glycerin or the like.

The preparations suitable for oral ingestion or administration includesadditives generally used in foods such as sweeteners, coloring agents,preservatives, thickening stabilizers, antioxidants, color developers,bleaching agents, fungicides, gum bases, bitter agents, enzymes, glazingagents, sour agents, seasonings, emulsifiers, enhancers, manufacturefacilitating agents, flavors, spice extracts and the like.

The product forms or preparations suitable for oral ingestion oradministration may be processed to, for example, tablets, powders,granules, pills, suspensions, emulsions, infusions/decoctions, capsules,drinks, liquids, elixirs, extracts, tinctures, or fluid extracts in aunit packaging form per ingestion, depending on ingestion period,ingestion frequency, ingestion dose and the like. For example, “unitpackaging form per ingestion” means that a pre-determined amount ispackaged for each ingestion, and “unit packaging form for 1 week to 3months” means that an ingestion dose for 1 week to 3 months is packaged.Examples of the unit packaging form include a form for prescribing apredetermined amount, such as a pack, a package, a bottle and the like.With respect to the “unit packaging form per ingestion”, for example, ifthe product form or preparation is a drink, 50 mg or more of citrullineor a salt thereof and citicoline or a salt thereof suspended ordissolved in a drink may be all packaged in a bottle for one ingestion.

With respect to the “unit packaging form for 1 week to 3 months”, forexample, it is ingested once a day to give a daily ingestion dose of 300mg, 50 mg of citrulline or a salt thereof and citicoline or a saltthereof are contained in a tablet, and 42 to 540 tablets are packaged ineach packaging form.

With respect to preparations suitable for parenteral administration, forexample, injections preferably comprise a sterilized aqueous preparationcontaining citrulline or a salt thereof and citicoline or a saltthereof, which is isotonic to the recipient's blood. In the case of aninjection, for example, a solution for injection is prepared using acarrier containing a salt solution, a glucose solution, or a mixture ofa salt solution and a glucose solution.

One or more of auxiliary components selected from the above-describedantiseptics, preservatives, excipients, disintegrating agents,lubricants, binders, surfactants, plasticizers and the like for oralpreparations can also be employed in these parenteral preparations.

The concentrations of citrulline or a salt thereof and citicoline or asalt thereof in the agent for preventing or improving decline in brainfunction of the present invention are appropriately determined accordingto the kind of the preparation, the effect expected by theadministration of the preparation or the like. Citrulline or a saltthereof is usually contained in an amount of 0.1 to 90% by weight,preferably 0.5 to 70% by weight, particularly preferably 1 to 50% byweight. Citicoline or a salt thereof is usually contained in an amountof 0.1 to 90% by weight, preferably 0.5 to 70% by weight, particularlypreferably 1 to 50% by weight.

The administration dose and administration frequency of the agent forpreventing or improving decline in brain function of the presentinvention vary depending on the administration form, the patient's ageand body weight, and the characteristics or severity of the symptoms tobe treated. Usually, the agent is administered once to several times perday in an amount to give a daily dose of 50 mg to 30 g, preferably 100mg to 10 g, particularly preferably 200 mg to 3 g per adult in terms ofcitrulline or a salt thereof. Further, usually, the agent isadministered once to several times per day in an amount to give a dailydose of 50 mg to 30 g, preferably 100 mg to 10 g, particularlypreferably 200 mg to 3 g per adult in terms of citicoline or a saltthereof.

Ingestion or administration period is not particularly limited, but itis generally 1 day to 1 year, preferably 1 week to 3 months.

The agent for preventing or improving decline in brain function of thepresent invention can be used for protecting brain neuronal cells. Theagent for preventing or improving decline in brain function of thepresent invention can be used for preventing or improving decline inbrain function that is developed from brain neuronal cell death.Examples of the effects expected by preventing or improving decline inbrain function includes prevention or improvement of decreasedperception ability, decreased memory learning ability, decreasedthinking ability, decreased concentration, decreased attention,decreased judgment ability, depression, decreased cognitive function anddecreased exercise performance caused thereby.

Therefore, the agent for preventing or improving decline in brainfunction of the present invention is administered to a subject in needof preventing occurrence of these symptoms or a subject having thesesymptoms, thereby preventing or improving the symptoms.

Further, in the present invention, citrulline or a salt thereof andciticoline or a salt thereof may be used in order to manufacture theagent for preventing or improving decline in brain function.

Further, the present invention includes a method for protecting brainneuronal cells. The method of the present invention comprises the stepof ingesting or administering citrulline or a salt thereof andciticoline or a salt thereof to a subject in need of protecting thebrain neuronal cells in amounts sufficient to protect the brain neuronalcells of the subject.

Further, the present invention includes a method for preventing orimproving decline in brain function. The method of the present inventionincludes the step of ingesting or administering citrulline or a saltthereof and citicoline or a salt thereof to a subject in need ofpreventing or improving decline in brain function, in amounts sufficientto prevent or improve decline in brain function of the subject.

Hereinafter, Experimental Examples regarding citrulline and citicolinefor preventing or improving decline in brain function will be described.

Experimental Example: 10-week-old male C57BL/6J mice (CLEA Japan, Inc.approximately 25 g of average body weight per mouse) were preliminarilyacclimated for 1 week, and provided for experiments. L-citrulline andciticoline were obtained from KYOWA HAKKO BIO CO., LTD.

C57BL/6J mice were divided into a sham-operation group (Group 1) and abilateral common carotid arteries occlusion group, which was performedunder halothane anesthesia for 20 minutes. One day after reperfusion,the bilateral common carotid arteries occlusion group was furtherdivided into a distilled water-treated group (Group 2), a low-doseL-citrulline-treated group (40 mg/kg: Group 3), a low-doseciticoline-treated group (40 mg/kg: Group 4), a high-doseL-citrulline-treated group (100 mg/kg: Group 5), a high-doseciticoline-treated group (100 mg/kg: Group 6), and a combinationtreatment group of a low dose of L-citrulline and a low dose ofciticoline (each 40 mg/kg: Group 7). Each sample was orally administeredfor 12 days. One week after starting administration, learningmemory-improving effects were examined by a Y-maze test, a passiveavoidance test, and a novel object recognition test. After evaluatinglearning memory functions, the hippocampus was removed and viability ofhippocampal nerve cells was evaluated.

Y-Maze Test

In the Y-maze test, a Y-maze apparatus with three arms (50×16×32 cm) wasused. Mice were placed at any one arm of the Y-maze and were allowed toexplore freely through the maze for 8 minutes. The sequence of armentries was recorded. The number of entries into each arm was countedduring the measurement time, which was defined as the total number ofentries. Of them, the successive entry into the three different arms wasexamined, and this number was defined as the spontaneous alternationbehavior number. The spontaneous alternation behavior number was dividedby (the total number of arm entries minus two), and then multiplied by100, which was defined as the spontaneous alternation behavior rate(Alternation) and used as an index for spontaneous alternation behavior.

As shown in FIG. 1 , a low Alternation was observed in Group 2, comparedto Group 1. Compared to Group 2, a high Alternation was observed inGroup 7 that is a combination group of Group 3 and Group 4, in each ofwhich Alternation cannot be improved. An improved effect of memorybehavior was recognized in Group 7, equivalent to those of Group 5 andGroup 6.

Passive Avoidance Test

The passive avoidance test was performed using a box consisting of dark(25×25×25 cm) and light compartments (14×10×25 cm) and being equippedwith an electrical stimulation apparatus at the bottom of the darkcompartment. During training, as soon as the mouse entered the darkcompartment from the light compartment, the door was closed, and anelectrical stimulation (0.3 mA, for 2 seconds) was delivered. Twentyfour hours after training, the mouse was placed again in the lightcompartment, and the latency time spent in the light compartment wasrecorded with a maximum latency of 300 seconds.

As shown in FIG. 2 , a significantly low latency time was observed inGroup 2, compared to Group 1. Compared to Group 2, a high latency timewas observed in Group 7 that is a combination group of Group 3 and Group4, in each of which the latency time cannot be improved. An improvedeffect of memory behavior was recognized in Group 7, equivalent to thoseof Group 5 and Group 6.

Novel Object Recognition Test

The novel object recognition test was performed after mice wereindividually acclimated to the open field apparatus (35×25×35 cm) for 2days. During training, mice were allowed to explore freely for 10minutes in an apparatus where two identical objects were symmetricallyplaced. After 1 hour, one object was replaced by a novel object, andmice were allowed to explore freely for 5 minutes. Object explorationwas defined as standing on the objects, touching the object with thenose, or sniffing the object within 1 cm from the object. A ratio of thefrequency of exploring the familiar or novel object over the totalexploration frequency was calculated, and used as an index for visualrecognition memory.

As shown in FIG. 3 , the increase in the frequency of exploring thenovel object observed in Group 1 was not observed in Group 2. Asignificant increase in the frequency of exploring the novel object wasobserved in Group 7 that is a combination group of Group 3 and Group 4,in each of which the frequency of exploring the novel object cannot beincreased. A remarkably improved effect of memory behavior wasrecognized in Group 7, equivalent to those of Group 5 and Group 6.

Evaluation of Viability of Hippocampal Nerve Cells

Twelve days after bilateral occlusion of the common carotid arteries,mice were anesthetized with pentobarbital sodium, and perfused withice-cold phosphate buffer (PBS, pH 7.4) until the blood in the body wascompletely removed. Immediately, mice were perfused with a fixativesolution containing 4% paraformaldehyde. The brain tissues were fixed at4° C. for 24 hours, and coronal sections having a thickness of 50 μmwere prepared. Serial sections containing the hippocampus were stainedwith propidium iodide (PI, 5 μmol/L) dissolved in PBS and observed undera fluorescent microscope. Surviving and non-surviving nerve cells in theCA1 pyramidal cell layer of the hippocampus were counted at 1.4 to 1.8mm posterior to bregma, and cells with PI-positive nuclei and typicalmorphological characteristics were defined as surviving nerve cells. Thecell viability was calculated from a ratio to the average number ofsurviving nerve cells in Group 1.

As shown in FIG. 4 , a significant reduction in the viability ofhippocampal neurons was observed in Group 2, compared to Group 1.Compared to Group 2, a significantly high viability of hippocampal nervecells was observed in Group 7 that is a combination group of Group 3 andGroup 4, in each of which the viability of hippocampal nerve cellscannot be improved. A nerve cell-protecting effect was recognized inGroup 7, equivalent to those of Group 5 and Group 6.

Taken together, it was clarified that the memory behavior can besynergistically and effectively improved by using citrulline incombination with citicoline, suggesting that the agent of the presentinvention is excellent in the prevention or improvement of decline inbrain function.

Hereinafter, Examples of the present invention will be described.

Example 1

Preparation of Tablet Containing Citrulline and Citicoline

120 kg of L-citrulline, 120 kg of citicoline, 19 kg of cyclicoligosaccharide, 57 kg of cellulose, and 1 kg of pullulan weregranulated using a fluid bed granulator dryer. The granulated productthus obtained and 3 kg of calcium stearate were mixed using a conicalblender, and compression-molded in a rotary compression molding machineto give a tablet.

Example 2

Preparation of Enteric Tablet Containing Citrulline and Citicoline

The surface of the tablet prepared in Example 1 was coated with ashellac solution to give an enteric tablet.

Example 3

Preparation of Enteric Capsule Containing Citrulline and Citicoline

120 kg of L-citrulline, 120 kg of citicoline, 19 kg of cyclicoligosaccharide, 57 kg of cellulose, 3 kg of calcium stearate and 1 kgof pullulan were mixed using a conical blender. 20 kg of the obtainedmixture and 0.2 kg of silicon dioxide were mixed and stirred. Theobtained mixture was fed into a capsule filling machine, and filled inhard capsules to give a hard capsule. The surface of the obtained hardcapsule was coated with a zein solution to give an enteric capsule.

Example 4

Preparation of Drink Containing Citrulline and Citicoline (1)

1.28 kg of L-citrulline, 1.28 kg of citicoline, 3 kg of erythritol, 0.05kg of citric acid, 3 g of artificial sweetener, and 0.06 kg of flavorwere dissolved in 50 L of water at 70° C. under stirring, and thesolution was adjusted to pH 3.3 with citric acid. The mixture wassterilized by plate sterilization and filled in a bottle. Then, thebottle is sterilized by pasteurizer to give a drink.

Example 5

Preparation of Drink Containing Citrulline and Citicoline (2)

20 mg of L-citrulline, 20 mg of citicoline, 20 mg of L-arginine, aproper amount of fructose glucose syrup, salt, citric acid, flavor, Nacitrate, Ca lactate, ferric pyrophosphate, Ca gluconate, K chloride, Mgchloride, and a sweetener were blended to give a 555 mL of drink.

Example 6

Preparation of Drink Containing Citrulline and Citicoline (3)

100 mg of L-citrulline, 100 mg of citicoline, 100 mg of L-arginine, 2.5mg of L-alanine, 2.5 mg of L-glycine, 2.5 mg of L-leucine, 1.3 mg ofL-isoleucine, 1.3 mg of L-valine and a proper amount of a flavor and asweetener were blended to give a 300 ml of drink.

The present application is based on Japanese patent Application No.2012-238542 filed on Oct. 30, 2012, the entire contents of which areincorporated hereinto by reference. All references cited herein areincorporated in their entirety.

INDUSTRIAL APPLICABILITY

According to the present invention, an agent for preventing or improvingdecline in brain function such as decreased perception ability,decreased memory learning ability, decreased thinking ability, decreasedconcentration, decreased attention, decreased judgment ability,depression, and decreased exercise performance caused thereby,comprising citrulline or a salt thereof and citicoline or a salt thereofas active ingredients is provided. According to the agent of the presentinvention, memory behavior can be synergistically and effectivelyimproved by using citrulline or a salt thereof in combination withciticoline or a salt thereof, and excellent effect as the agent forpreventing or improving decline in brain function can be obtained.

The invention claimed is:
 1. A method of protecting brain neuronal cellscomprising orally administering an effective amount of the combinationof (a) citrulline or a salt thereof and (b) citicoline or a salt thereofto a human subject in need thereof, thereby protecting brain neuronalcells in the subject, wherein citrulline or a salt thereof andciticoline or a salt thereof are each orally administered at a dose of100-300 mg/day.
 2. The method of claim 1, wherein citrulline or a saltthereof and citicoline or a salt thereof are each orally administered ata dose of 300 mg/day.
 3. The method of claim 1, wherein citrulline or asalt thereof and citicoline or a salt thereof are each orallyadministered at a dose of 200-300 mg/day.
 4. The method of claim 1,wherein memory behavior is improved in the subject.
 5. The method ofclaim 1, wherein visual recognition memory is improved in the subject.6. The method of claim 1, wherein hippocampal nerve cells are protectedin the subject.
 7. The method of claim 1, wherein the method does notinclude surgery, therapy, or diagnosis of the subject by a medicaldoctor.
 8. The method of claim 1, wherein the subject suffers fromdepression or has one or more impairments in perception ability, memorylearning ability, thinking ability, concentration, attention, judgmentability, and exercise performance caused by a decline in brain function.9. The method of claim 2, wherein memory behavior is improved in thesubject.
 10. The method of claim 2, wherein visual recognition memory isimproved in the subject.
 11. The method of claim 2, wherein hippocampalnerve cells are protected in the subject.
 12. The method of claim 2,wherein the method does not include surgery, therapy, or diagnosis ofthe subject by a medical doctor.
 13. The method of claim 2, wherein thesubject suffers from depression or has one or more impairments inperception ability, memory learning ability, thinking ability,concentration, attention, judgment ability, and exercise performancecaused by a decline in brain function.
 14. The method of claim 3,wherein memory behavior is improved in the subject.
 15. The method ofclaim 3, wherein visual recognition memory is improved in the subject.16. The method of claim 3, wherein hippocampal nerve cells are protectedin the subject.
 17. The method of claim 3, wherein the method does notinclude surgery, therapy, or diagnosis of the subject by a medicaldoctor.
 18. The method of claim 3, wherein the subject suffers fromdepression or has one or more impairments in perception ability, memorylearning ability, thinking ability, concentration, attention, judgmentability, and exercise performance caused by a decline in brain function.